The field of this invention is proteins involved in transcription factor activation.
Cytokines trigger changes in gene expression by modifying the activity of otherwise latent transcription factors (Hill and Treisman, 1995). Nuclear factor xcexaB (NF-xcexaB) is a prominent example of how such an external stimulus is converted into an active transcription factor (Verma et al., 1995). The NF-xcexaB system is composed of homo- and heterodimers of members of the Rel family of related transcription factors that control the expression of numerous immune and inflammatory response genes as well as important viral genes (Lenardo and Baltimore, 1989; Baeuerle and Henkel, 1994). The activity of NF-xcexaB transcription factors is regulated by their subcellular localization (Verma et al., 1995). Inmost cell types, NF-xcexaB is present as a heterodimer comprising of a 50 kDa and a 65 kDa subunit. This heterodimer is sequestered in the cytoplasm in association with IxcexaBxcex1 a member of the IxcexaB family of inhibitory proteins (Finco and Baldwin, 1995; Thanos and Maniatis, 1995; Verma et al., 1995). IxcexaBxcex1 masks the nuclear localization signal of NF-xcexaB and thereby prevents NF-xcexaB nuclear translocation. Conversion of NF-xcexaB into an active transcription factor that translocates into the nucleus and binds to cognate DNA sequences requires the phosphorylation and subsequent ubiquitin-dependent degradation of IxcexaBxcex1 in the 26s proteasome. Signal-induced phosphorylation of IxcexaBxcex1 occurs at serines 32 and 36. Mutation of one or both of these serines renders IxcexaBxcex1 resistant to ubiquitination and proteolytic degradation (Chen et al., 1995).
The pleiotropic cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) are among the physiological inducers of IxcexaB phosphorylation and subsequent NF-xcexaB activation (Osborn et al., 1989; Beg et al., 1993). Although TNF and IL-1 initiate signaling cascades leading to NF-xcexaB activation via distinct families of cell-surface receptors (Smith et al., 1994; Dinarello, 1996), both pathways utilize members of the TNF receptor-associated factor (TRAF) family of adaptor proteins as signal transducers (Rothe et al., 1995; Hsu et al., 1996; Cao et al., 1996b). TRAF proteins were originally found to associate directly with the cytoplasmic domains of several members of the TNF receptor family including the 75 kDa TNF receptor (TNFR2), CD40, CD30, and the lymphotoxin-xcex2 receptor (Rothe et al., 1994; Hu et al., 1994; Cheng et al., 1995; Mosialos et al., 1995; Song and Donner, 1995; Sato et al., 1995; Lee et al., 1996; Gedrich et al., 1996; Ansieau et al., 1996). In addition, TRAF proteins are recruited indirectly to the 55 kDa TNF receptor (TNFR1) by the adaptor protein TRADD (Hsu et al., 1996). Activation of NF-xcexaB by TNF requires TRAF2 (Rothe et al., 1995; Hsu et al., 1996). TRAF5 has also been implicated in NF-xcexaB activation by members of the TNF receptor family (Nakano et al., 1996). In contrast, TRAF6 participates in NF-xcexaB activation by IL-1 (Cao et al., 1996b). Upon IL-1 treatment, TRAF6 associates with IRAK, a serine-threonine kinase that binds to the IL-1 receptor complex (Cao et al., 1996a).
The NF-xcexaB-inducing kinase (NIK) is a member of the MAP kinase kinase kinase (MAP3K) family that was identified as a TRAF2-interacting protein (Malinin et al., 1997). NIK activates NF-xcexaB when overexpressed, and kinase-inactive mutants of NIK comprising its TRAF2-interacting C-terminal domain (NIK(624-947)) or lacking two crucial lysine residues in its kinase domain (NIK(KK429-430AA)) behave as dominant-negative inhibitors that suppress TNF-, IL-1-, and TRAF2-induced NF-xcexaB activation (Malinin et al., 1997). Recently, NIK was found to associate with additional members of the TRAF family, including TRAF5 and TRAF6. Catalytically inactive mutants of NIK also inhibited TRAF5- and TRAF6-induced NF-xcexaB activation, thus providing a unifying concept for NIK as a common mediator in the NF-xcexaB signaling cascades triggered by TNF and IL-1 downstream of TRAFs.
Here, we disclose a novel human kinase IxcexaB Kinase, IKK-xcex1, as a NIK-interacting protein. IKK-xcex1 has sequence similarity to the conceptual translate of a previously identified open reading frame (SEQ ID NO:5) postulated to encode a serine-threonine kinase of unknown function (xe2x80x98Conserved Helix-loop-helix Ubiquitous Kinasexe2x80x99 or CHUK, Connelly and Marcu, 1995; Mock et al., 1995). Catalytically inactive mutants of IKK-xcex1 are shown to suppress NF-xcexaB activation induced by TNF and IL-1 stimulation as well as by TRAF and NIK overexpression; transiently expressed IKK-xcex1 is shown to associate with the endogenous IxcexaBxcex1 complex; and IKK-xcex1 is shown to phosphorylate IxcexaBxcex1 on serines 32 and 36.
The invention provides methods and compositions relating to isolated IKK-xcex1 polypeptides, related nucleic acids, polypeptide domains thereof having IKK-xcex1-specific structure and activity and modulators of IKK-xcex1 function, particularly IxcexaB kinase activity. IKK-xcex1 polypeptides can regulate NFxcexaB activation and hence provide important regulators of cell function. The polypeptides may be produced recombinantly from transformed host cells from the subject IKK-xcex1 polypeptide encoding nucleic acids or purified from mammalian cells. The invention provides isolated IKK-xcex1 hybridization probes and primers capable of specifically hybridizing with the disclosed IKK-xcex1 gene, IKK-xcex1-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for IKK-xcex1 transcripts), therapy (e.g. IKK-xcex1 kinase inhibitors to inhibit TNF signal transduction) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating other transcriptional regulators, reagents for screening chemical libraries for lead pharmacological agents, etc.).
The nucleotide sequence of a natural cDNA encoding a human IKK-xcex1 polypeptide is shown as SEQ ID NO:3, and the full conceptual translate is shown as SEQ ID NO:4. The IKK-xcex1 polypeptides of the invention include incomplete translates of SEQ ID NO:3, particularly of SEQ ID NO:3, residues 1-638, which translates and deletion mutants of SEQ ID NO:4 have human IKK-xcex1-specific amino acid sequence, binding specificity or function and comprise at least one of Cys30, Leu403, Glu543, Leu604, Thr679, Ser680, Pro684, Thr686 and Ser687. Preferred translates/deletion mutants comprise at least a 6, preferably at least a 12, more preferably at least an 18 residue Cys30, Leu403, Glu543, Leu604, Thr679, Ser680, Pro684, Thr686 or Ser687-containing domain of SEQ ID NO:4, preferably including at least 8, more preferably at least 12, most preferably at least 20 contiguous residues which immediately flank said residue on one, preferably both sides, with said residue preferably residing within said contigous residues, see, e.g. Table 1A, which mutants provide hIKK-xcex1 specific epitopes and immunogens.
TABLE 1A. Exemplary IKK-xcex1 polypeptides having IKK-xcex1 binding specificity
xcex1xcex941(SEQ ID NO:4, residues 1-30)
xcex1xcex942(SEQ ID NO:4, residues 22-31)
xcex1xcex943(SEQ ID NO:4, residues 599-608)
xcex1xcex944(SEQ ID NO:4, residues 601-681)
xcex1xcex945(SEQ ID NO:4, residues 604-679)
xcex1xcex946(SEQ ID NO:4, residues 670-687)
xcex1xcex947(SEQ ID NO:4, residues 679-687)
xcex1xcex948(SEQ ID NO:4, residues 680-690)
xcex1xcex949(SEQ ID NO:4, residues 684-695)
xcex1xcex9410(SEQ ID NO:4, residues 686-699)
xcex1xcex9411(SEQ ID NO:4, residues 312-345)
xcex1xcex9412(SEQ ID NO:4, residues 419-444)
xcex1xcex9413(SEQ ID NO:4, residues 495-503)
xcex1xcex9414(SEQ ID NO:4, residues 565-590)
xcex1xcex9415(SEQ ID NO:4, residues 610-627)
xcex1xcex9416(SEQ ID NO:4, residues 627-638)
xcex1xcex9417(SEQ ID NO:4, residues 715-740)
xcex1xcex9418(SEQ ID NO:4, residues 737-745)
In a particular embodiment, the invention provides IKK-xcex1Glu543 polypeptides, IKK-xcex1Glu543 polypeptide-encoding nucleic acids/polynucleotides, and IKK-xcex1Glu543 polypeptide-based methods (below), which IKK-xcex1Glu543 polypeptides comprise at least 8, preferably at least 10, more preferably at least 12, more preferably at least 16, most preferably at least 24 consecutive amino acid residues of the amino acid sequence set forth as SEQ ID NO:4, which consecutive amino acid residues comprise the amino acid residue 543 (Glu) of SEQ ID NO:4. Exemplary IKK-xcex1Glu543 polypeptides having IKK-xcex1Glu543 binding specificity and immunologically distinguishable from IKK-xcex1Gly543 are shown in Table 1B.
TABLE 1B. Exemplary IKK-xcex1Glu543 polypeptides having IKK-xcex1Glu543 binding specificity
xcex1xcex9419(SEQ ID NO:4, residues 540-548)
xcex1xcex9420(SEQ ID NO:4, residues 543-550)
xcex1xcex9421(SEQ ID NO:4, residues 536-543)
xcex1xcex9422(SEQ ID NO:4, residues 534-554)
xcex1xcex9423(SEQ ID NO:4, residues 533-543)
xcex1xcex9424(SEQ ID NO:4, residues 543-563)
xcex1xcex9425(SEQ ID NO:4, residues 542-549)
xcex1xcex9426(SEQ ID NO:4, residues 538-545)
xcex1xcex9427(SEQ ID NO:4, residues 541-547)
xcex1xcex9428(SEQ ID NO:4, residues 403-543)
xcex1xcex9429(SEQ ID NO:4, residues 403-604)
xcex1xcex9430(SEQ ID NO:4, residues 403-679)
xcex1xcex9431(SEQ ID NO:4, residues 403-680)
xcex1xcex9432(SEQ ID NO:4, residues 403-687)
xcex1xcex9433(SEQ ID NO:4, residues 543-604)
xcex1xcex9434(SEQ ID NO:4, residues 543-679)
xcex1xcex9435(SEQ ID NO:4, residues 543-684)
xcex1xcex9436(SEQ ID NO:4, residues 543-687)
The subject domains provide IKK-xcex1 domain specific activity or function, such as IKK-xcex1-specific kinase or kinase inhibitory activity, NIK-binding or binding inhibitory activity, IxcexaB-binding or binding inhibitory activity, NFxcexaB activating or inhibitory activity or antibody binding. Preferred domains phosphorylate at least one and preferably both the serine 32 and 36 of IxcexaB (Verma, I. M., et al. (1995)). As used herein, Ser32 and Ser36 of IxcexaB refers collectively to the two serine residues which are part of the consensus sequence DSGL/IXSM/L (e.g. ser 32 and 36 in IxcexaBxcex1, ser 19 and 23 in IxcexaBxcex2, and ser 157 and 161, or 18 and 22, depending on the usage of methionines, in IxcexaBxcex5, respectively.
IKK-xcex1-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of an IKK-xcex1 polypeptide with a binding target is evaluated. The binding target may be a natural intracellular binding target such as an IKK-xcex1 substrate, a IKK-xcex1 regulating protein or other regulator that directly modulates IKK-xcex1 activity or its localization; or non-natural binding target such a specific immune protein such as an antibody, or an IKK-xcex1 specific agent such as those identified in screening assays such as described below. IKK-xcex1-binding specificity may assayed by kinase activity or binding equilibrium constants (usually at least about 107Mxe2x88x921, preferably at least about 108Mxe2x88x921, more preferably at least about 109Mxe2x88x921), by the ability of the subject polypeptide to function as negative mutants in IKK-xcex1-expressing cells, to elicit IKK-xcex1 specific antibody in a heterologous host (e.g. a rodent or rabbit), etc. In any event, the IKK-xcex1 binding specificity of the subject IKK-xcex1 polypeptides necessarily distinguishes the murine and human CHUK sequences of Connelly and Marcu (1995) as well as IKK-xcex2 (SEQ ID NO:4).
The claimed IKK-xcex1 polypeptides are isolated or pure: an xe2x80x9cisolatedxe2x80x9d polypeptide is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total polypeptide in a given sample and a pure polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample. In a particular embodiments, IKK-xcex1 polypeptides are isolated from a MKP-1 precipitable complex, isolated from a IKK complex, and/or isolated from IKK-xcex2. The IKK-xcex1 polypeptides and polypeptide domains may be synthesized, produced by recombinant technology, or purified from mammalian, preferably human cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art.
The invention provides binding agents specific to IKK polypeptides, preferably the claimed IKK-xcex1 polypeptides, including substrates, agonists, antagonists, natural intracellular binding targets, etc., methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, specific binding agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with improper utilization of a pathway involving the subject proteins, e.g. NF-xcexaB activation. Novel IKK-specific binding agents include IKK-specific receptors, such as somatically recombined polypeptide receptors like specific antibodies or T-cell antigen receptors (see, e.g. Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural intracellular binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries such as described below, etc. Agents of particular interest modulate IKK function, e.g. IKK-dependent transcriptional activation. For example, a wide variety of inhibitors of IKK IxcexaB kinase activity may be used to regulate signal transduction involving IxcexaB. Exemplary IKK IxcexaB kinase inhibitors include known classes of serine/threonine kinase (e.g. PKC) inhibitors such as competitive inhibitors of ATP and substrate binding, antibiotics, IKK-derived peptide inhibitors, etc., see Tables II and III. IKK specificity and activity are readily quantified in high throughput kinase assays using panels of protein kinases (see cited references and Examples).
Preferred inhibitors include natural compounds such as staurosporine (Omura S, et al. J Antibiot (Tokyo) 1995 July;48(7):535-48), produced by a marine organism, and synthetic compounds such as PD 153035, which also potently inhibits the EGF receptor protein kinase (Fry DW et al. Science 1994 August 19;265(5175):1093-5). Members of the tyrphostin family of synthetic protein kinase inhibitors are also useful; these include compounds which are pure ATP competitors, compounds which are pure substrate competitors, and compounds which are mixed competitors: compete with both ATP and substrate (Levitzki A and Gazit A, Science 1995 March 24;267(5205):1782-8). Additional IKK inhibitors include peptide-based substrate competitors endogenously made by the mammalian cell, e.g. PKI (protein kinase inhibitor, Seasholtz AF et al., Proc Natl Acad Sci USA 1995 February 28;92(5):1734-8), or proteins inhibiting cdc kinases (Correa-Bordes J and Nurse P, Cell 1995 December 15;83(6):1001-9). Additional small peptide based substrate competitive kinase inhibitors and allosteric inhibitors (inhibitory mechanisms independent of ATP or substrate competition) are readily generated by established methods (Hvalby O, et al. Proc Natl Acad Sci USA 1994 May 24;91(11):4761-5; Barja P, et al., Cell Immunol 1994 January;153(1):28-38; Villar-Palasi C, Biochim Biophys Acta 1994 December 30;1224(3):384-8; Liu W Z, et al., Biochemistry 1994 August 23;33(33):10120-6).
Accordingly, the invention provides methods for modulating signal transduction involving IxcexaB in a cell comprising the step of modulating IKK kinase activity, e.g. by contacting the cell with a serine/threonine kinase inhibitor. The cell may reside in culture or in situ, i.e. within the natural host. Preferred inhibitors are orally active in mammalian hosts. For diagnostic uses, the inhibitors or other IKK binding agents are frequently labeled, such as with fluorescent, radioactive, chemiluminescent, or other easily detectable molecules, either conjugated directly to the binding agent or conjugated to a probe specific for the binding agent.
The amino acid sequences of the disclosed IKK-xcex1 polypeptides are used to back-translate IKK-xcex1 polypeptide-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural IKK-xcex1-encoding nucleic acid sequences (xe2x80x9cGCGxe2x80x9d software, Genetics Computer Group, Inc, Madison Wis.). IKK-xcex1-encoding nucleic acids used in IKK-xcex1-expression vectors and incorporated into recombinant host cells, e.g. for expression and screening, transgenic animals, e.g. for functional studies such as the efficacy of candidate drugs for disease associated with IKK-xcex1-modulated cell function, etc.
The invention also provides nucleic acid hybridization probes and replication/amplification primers having a IKK-xcex1 cDNA specific sequence comprising at least 12, preferably at least 24, more preferably at least 36 and most preferably at least contiguous 96 bases of a strand of SEQ ID NO:3, particularly of SEQ ID NO:2, nucleotides 1-1913, and preferably including at least one of bases 1-92, 1811, 1812, 1992, 1995, 2034, 2035, 2039, 2040, 2050, 2055 and 2060, and sufficient to specifically hybridize with a second nucleic acid comprising the complementary strand of SEQ ID NO:3 in the presence of a third nucleic acid comprising (SEQ ID NO:5). Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5xc3x97SSPE (0.18 M NaCl, 0.01 M NaPO4, pH7.7, 0.001 M EDTA) buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5xc3x97SSPE buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE buffer at 42xc2x0 C. IKK-xcex1 nucleic acids can also be distinguished using alignment algorithms, such as BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410).
In a particular embodiment, the invention provides IKK-xcex1A1628 polynucleotides, comprising at least 18, 24, 36, 48, 72, 148, 356 or 728 consecutive nucleotides of the nucleotide sequence set forth as SEQ ID NO:3, which consecutive polynucleotides comprise the polynucleotide 1628 (A) of SEQ ID NO:3. Exemplary IKK-xcex1A1628 polynucleotides and allele specific oligonucleotide probes having IKK-xcex1Glu543 binding specificity and distinguishable by hybridization assays from IKKxcex1G1628 are shown in Table IV.
The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Recombinant nucleic acids comprising the nucleotide sequence of SEQ ID NO:3, or requisite fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by (i.e. contiguous with) a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.
The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of IKK-xcex1 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional IKK-xcex1 homologs and structural analogs. In diagnosis, IKK-xcex1 hybridization probes find use in identifying wild-type and mutant IKK-xcex1 alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic IKK-xcex1 nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active IKK-xcex1.
The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of a IKK modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate IKK interaction with a natural IKK binding target, in particular, IKK phosphorylation of IxcexaB-derived substrates, particularly IxcexaB and NIK substrates. A wide variety of assays for binding agents are provided including labeled in vitro protein-protein binding assays, immunoassays, cell based assays, etc. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
In vitro binding assays employ a mixture of components including an IKK polypeptide, which may be part of a fusion product with another peptide or polypeptide, e.g. a tag for detection or anchoring, etc. The assay mixtures comprise a natural intracellular IKK binding target. In a particular embodiment, the binding target is a substrate comprising IxcexaB serines 32 and/or 36. Such substrates comprise a IxcexaBxcex1, xcex2 or xcex5 peptide including the serine 32 and/or 36 residue and at least 5, preferably at least 10, and more preferably at least 20 naturally occurring immediately flanking residues on each side (e.g. for serine 36 peptides, residues 26-46, 22-42, or 12-32 or 151-171 for IxcexaBxcex1, xcex2 or xcex5-derived substrates, respectively). While native full-length binding targets may be used, it is frequently preferred to use portions (e.g. peptides) thereof so long as the portion provides binding affinity and avidity to the subject IKK polypeptide conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like ATP or ATP analogs (for kinase assays), salts, buffers, neutral proteins, e.g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.
The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the IKK polypeptide specifically binds the cellular binding target, portion or analog with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening.
After incubation, the agent-biased binding between the IKK polypeptide and one or more binding targets is detected by any convenient way. For IKK kinase assays, xe2x80x98bindingxe2x80x99 is generally detected by a change in the phosphorylation of a IKK-xcex1 substrate. In this embodiment, kinase activity may quantified by the transfer to the substrate of a labeled phosphate, where the label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, etc. A variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g. through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc.
A difference in the binding affinity of the IKK polypeptide to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the IKK polypeptide to the IKK binding target. Analogously, in the cell-based assay also described below, a difference in IKK-xcex1-dependent transcriptional activation in the presence and absence of an agent indicates the agent modulates IKK function. A difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.